Early diagnosis is a priority and highly desirable objective in all fields of medicament, particularly because it allows an appreciable improvement in the patient's life and a concomitant saving on the part of health care systems or on the part of the actual patients. In the particular case of the invention described herein, early diagnosis is that of potential or existing Toxoplasma gondii infection in pregnant women, with particular concern for the health of the foetus, and in infected subjects, particularly those with impaired immunity.
Toxoplasma gondii is an obligate intracellular parasite that infects all mammalian cells, including those of human subjects (McCabe and Remington, N. Engl. J. Med. 1988, 318-313-5). Morphologically, the parasite exhibits three distinct forms of infection: tachyzoite (asexual), bradyzoite (in tissue cysts, asexual) and sporozoite (in oocysts, sexual reproduction). Transmission typically occurs through ingestion of undercooked meat harbouring tissue cysts or vegetables contaminated with oocysts shed by cats. Human infection is generally asymptomatic and self-limiting in immunocompetent hosts. In contrast, in subjects with impaired immunity (particularly those affected by AIDS), toxoplasmosis is a severe opportunist infection, which may give rise to encephalitis with very serious outcomes (Luft, B. J., Remington J. S., 1992, Clin. Infect. Dis. 15, 211-22). Moreover, contracting primary infection during pregnancy may lead to miscarriages or to severe foetal disease in mammals.
For an extensive overview of the problem of toxoplasmosis the reader is, referred to the specific medical literature.
Diagnosis of T. gondii infection is established by isolating the micro-organism in the blood or body fluids, identifying the parasite in tissues, detecting specific nucleotide sequences with PCR, or detecting specific anti-T. gondii immunoglobulins produced by the host in response to the infection (Beaman et al., 1995 Principles and Practice of Infectious Diseases 4th Ed., Churchill Livingstone Inc., New York, 2455-75; Remington J S et al. 1995, Infectious Diseases of the Fetus and Newborn Infant, W. B. Saunders, Philadelphia, Pa., 140-267).
Main challenges for clinicians are the diagnosis of primary T. gondii infections in pregnant women and the diagnosis of congenital infection in their newborns/infants. In both cases, to implement suitable therapies in good time and to avoid possible damage to the foetus and newborns/infants, it is very important to establish if the parasitic infection has been contracted before or after conception in pregnant women. Moreover, it is essential determining when the vertical transmission from the mother to the foetus occurred. Finally, for the clinical management of newborns/infants there is an urgent need of a sensitive diagnostic method than can discriminate, early in their life, between infected and uninfected subjects, both born to mothers with primary toxoplasmosis in pregnancy.
Seroconversion during gestation and diagnosis of congenital infection in neonates are generally done by attempting to detect the presence of the various classes of anti-Toxoplasma immunoglobulins (IgG, IgM, IgA, avidity of IgG), and to compare the immunological profiles of the mother versus her child. However, the available commercial assays do not provide enough sensitivity and specificity to allow a correct diagnosis of infection in all patients. Therefore the availability of specific, sensitive and innovative diagnostic agents is desirable.
T. gondii antigens have long been known and available, first of all as antigen mixtures obtained in various ways (FR 2,226,468, Mérieux; SU 533376, Veterinary Research Institute; JP 54044016, Nihon Toketsu Kanso), then as subsequent isolations of pure antigens (EP 0 082 745, Mérieux; EP 0 301 961, INSERM, Pasteur; WO 89/5658, Transgene) and their characterization both as proteins, and of their respective genes (WO 89/08700, U. Leland, Dartmouth Coll.; U.S. Pat. No. 4,877,726, Res. Inst. Palo Alto; WO 89/12683, INSERM, Pasteur; EP 0 391 319, Mochida Pharm.; IT 1,196,817, CNR; EP 0 431 541, Behringwerke; WO 92/01067, CNRS; WO 92/02624, U. Flinders; WO 92/11366, Innogenetics, Smithkline Beecham; U.S. Pat. No. 5,215,917, Res. Inst. Palo Alto; WO 92/25689, FR 2702491, INSERM, Pasteur; WO 96/02654, bioMeriéux, Transgene; EP 0 710 724 Akzo; EP 0 724 016, bioMeriéux; EP 0 751 147, Behringwerke; U.S. Pat. No. 5,633,139, Res. Inst. Palo Alto; WO 97/27300, Innogenetics; U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst. Palo Alto; WO 99/32633, Heska; JP 11225783, Yano; WO 99/61906, Abbott; WO 99/66043, Smithkline Beecham; JP 2000300278, Yano; WO 00/164243, Virsol), and finally, the isolation and characterization of the antigenic regions of Toxoplasma gene products (WO 03/080839, Kenton S.r.l.) Numerous studies have found various different antigenic proteins of T. gondii and the gene sequences of these have also been determined.
Among the most interesting proteins both for diagnostic and therapeutic purposes, in the form of vaccines, we should mention: the microneme proteins (WO 03/080839, Kenton S.r.l.; Beghetto et al., The Journal of Infectious Diseases, 2005, 191:637-645; Beghetto et al., International Journal for Parasitology, 2003, 33:163-173); the surface antigens SAG1 (or P30) (WO 89/08700, Stanford University; WO 89712683 Pasteur, INSERM; WO 94/17813, WO 96/02654, Transgene, bioMeriéux; EP 0 724 016, WO 99/61906, U.S. Pat. No. 5,962,654, Harning et al., Clinical and Diagnostic Laboratory Immunology, May 1996, 355-357) and SAG2 (or P22) (Parmley et al., 1992, J. Clin. Microbiol. 30, 1127-33); the dense granule proteins GRA1 (or P24) (EP 0 301 961, Pasteur, INSERM; WO 89/05658, Transgene, Cesbron-Delauw, et al., 1989 P.N.A.S. USA 86, 7537-41), GRA2 (or P28) (WO 93/25689, INSERM, Pasteur; U.S. Pat. Nos. 5,633,139, 5,665,542, 5,686,575, Res. Inst. Palo Alto; Prince et al., Mol. Biochem. Parasitol., 34 3-14), GRA4 (Mevelec et al., Mol. Biochem. Parasitol. 56, 227-38), GRA6 (or P32) (FR 2,702,491, INSERM, Pasteur; Lecordier al., Mol. Biochem. Parasitol. 70, 85-94), GRA7 (WO 99/61906, Abbott; Jacobs et al., Mol. Biochem. Parasitol. 91, 237-49) and GRA3 (Robben et al. 2002, J. Biol. Chem. 277, 17544-47): the rhoptry antigens ROP1 (or P66) (U.S. Pat. No. 5,976,553, U. Leland; EP 0 431 541, Innogenetics) and ROP2 (or P54) (Sharma et al., J. Immunol., 131, 377-83).
As described in the above-mentioned references, the antigens were obtained with recombinant cDNA techniques in expression vectors. For example, for the antigen SAG1, WO 98/08700 uses known expression vectors in phage lambda-gt11. WO 98/12683 uses the same phage and transfects E. coli with a proprietary plasmid, or by preparing a special expression cassette, as in WO 96/02654. EP 0 724 016 obtains mimotopes using combinatorial expression libraries of peptides. EP 0 301 961 describes how to obtain excretion-secretion antigens with molecular weights ranging from 20 kDa to 185 kDa. WO 89/05658 describes a protein containing the epitopes of the 24 kDa protein recognized by the antibodies produced against Toxoplasma excretion-secretion antigens; this protein is obtained by transfection of cells by means of expression vectors. WO 03/080839 describes a method based on phage-display technology for identifying antigen fragments of T. gondii proteins and their use as diagnostic and immunogenic agents. The antigen P28 (GRA2) is described in U.S. Pat. No. 5,633,139 and the method of obtaining it is again implemented through expression in phage lambda-gt11. The antigen P32 (GRA6) is described in patent FR 2,702,491, the antigen ROP1 (P66) in U.S. Pat. No. 5,976,553, P35 (or GRA8) in EP 0 431 541, WO 99/57295 and WO 99/61906, and lastly P68 in EP 0 431 541.
Yang et al. (Parasitol. Res., 2004, 92: 58-64) describe a chimeric protein containing SAG1 and SAG2 and its use to develop immunity against T. gondii in mice.
Chinese Patent 11 94991C discloses a recombinant fusion protein containing two toxoplasma antigens (GRA6 and p30). No data are reported to show that assays based on this recombinant fusion protein display the required sensitivity in IgG- and IgM-based tests.
During the last ten years, several studies have reported the use of recombinant antigens for the serological diagnosis of T. gondii infection. Nevertheless, although promising none of the assays based on recombinant antigens displayed all the characteristics required to replace the tachyzoite antigen in IgG- and IgM-based tests, indicating that further work is needed before an immunoassay employing recombinant products will be available for clinical purposes.
Thus the main aim of the studies in this filed is to improve the performance of enzyme-linked immunoassays based on recombinant products, thus improving, for example early diagnosis of congenital toxoplasmosis in newborns/infants.